Efforts have been continued to identify and characterize those constituents responsible for the biological activities of the etiologic agent of Rocky Mountain spotted fever, Rickettsia rickettsii. Studies with various kinds of extracts suggested that the 120 K surface protein was the principal protective antigen. The 120 K protein-rich fractions from affinity columns stimulated in mice much better active immunity than did the original extract. The 120 K protein was heat labile; it was detected in immunoblots with monoclonal antibodies after exposure to temperatures of 25 degrees C but not 37 degrees C. Intact rickettsiae held at 56 degrees C for 15 min did not induce protective immunity in mice. It was shown by crossed radioimmunoelectrophoresis and immunoblotting with monoclonal antibodies that R. rickettsii LPS, erythrocyte-sensitizing substance (ESS), and the complement fixing (CF) antigen were all derivatives of the same rickettsial antigen. Though the 120 and 155 K proteins of R. rickettsii strains of high and low virulence for guinea pigs had identical relative mobilities and common epitopes, these proteins were structurally different. The same number of rickettsiae of high and low virulence strains was required to kill mice in less than 24 h, so it does not appear that the ability of rickettsiae to kill mice in this type of assay is a major factor in rickettsial virulence for guinea pigs and man. Guinea pigs inoculated with a low virulence strain, the Iowa strain, exhibited few or no symptoms of disease but were subsequently immune to challenge with a high dose of the virulent R strain. SDS-PAGE analysis revealed that the Iowa strain lacked a 32 K heat-modifiable protein found in other strains and had about 140 K protein instead of the 120 K protein described above. It is not presently known whether these changes in protein composition are related to the loss of virulence of the Iowa strain.